ApoE, a 34,000 molecular weight protein is the product of a single gene on chromosome 19 and exists in three major isoforms designated apoE2, apoE3 and apoE4 for review, see Mahley in: Molecular and Genetic Bases of Neurological Disease 2nd ed.; and Mahley (1988) Science 240:622-630. The different isoforms result from amino acid substitutions at amino acid residue positions 112 and 158. The common isoform, apoE3, has a cysteine residue at position 112 and an arginine residue at position 158. The apoE4 isoform differs from apoE3 only at position 112, which is an arginine residue. The apoE2 isoform, associated with type III hyperlipoproteinemia (Mahley (1988)), differs from apoE3 only at position 158, which is a cysteine residue. ApoE3 and apoE4 bind normally to the low density lipoprotein (LDL) receptor, whereas apoE2 does not.
ApoE contains two structural domains: an amino-terminal and a carboxy-terminal domain. Weisgraber (1994) Adv. Protein Chem. 45:249-302. Each domain is associated with a specific function. The amino terminal domain contains the lipoprotein receptor binding region and the carboxy-terminal domain contains the major lipid-binding elements. The two domains appear to interact with each other in an isoform-specific manner such that amino acid substitutions in one domain influence the function of the other domain, a phenomenon referred to as domain interaction. Domain interaction is responsible for the preference of apoE4 for very low density lipoproteins (VLDL) contrasted with the preference of apoE3 for high density lipoproteins (HDL). The specific amino acid residues in apoE4 that are involved in this interaction have been identified: arginine-61 in the amino-terminal domain and glutamic acid-255 in the carboxy-terminal domain. Dong et al. (1994) J. Biol. Chem. 269:22358-22365; and Dong and Weisgraber (1996) J. Biol. Chem. 271:19053-19057.
By redistributing lipids among the cells of different organs, apoE plays a critical role in lipid metabolism. While apoE exerts this global transport mechanism in chylomicron and VLDL metabolism, it also functions in the local transport of lipids among cells within a tissue. Cells with excess cholesterol and other lipids may release these substances to apoE-lipid complexes or to HDL containing apoE, which can transport the lipids to cells requiring them for proliferation or repair. The apoE on these lipoprotein particles mediates their interaction and uptake via the LDL receptor or the LRP.
ApoE plays a neurobiological role. ApoE mRNA is abundant in the brain, where it is synthesized and secreted primarily by astrocytes. Elshourbagy et al. (1985) Proc. Natl. Acad. Sci. USA 82:203-207; Boyles et al. (1985) J. Clin. Invest. 76:1501-1513; and Pitas et al. (1987) Biochem. Biophys. Acta 917:148-161. The brain is second only to the liver in the level of apoE mRNA expression. ApoE-containing lipoproteins are found in the cerebrospinal fluid and appear to play a major role in lipid transport in the central nervous system (CNS). Pitas et al. (1987) J. Biol. Chem. 262:14352-14360. In fact, the major cerebrospinal fluid lipoprotein is an apoE-containing HDL. ApoE plus a source of lipid promotes marked neurite extension in dorsal root ganglion cells in culture. Handelmann et al. (1992) J. Lipid Res. 33:1677-1688. ApoE levels dramatically increase (about 250-fold) after peripheral nerve injury. Müller et al. (1985) Science 228:499-501; and Ignatius et al. (1986) Proc. Natl. Acad. Sci. USA 83:1125-1129. ApoE appears to participate both in the scavenging of lipids generated after axon degeneration and in the redistribution of these lipids to sprouting neurites for axon regeneration and later to Schwann cells for remyelination of the new axons. Boyles et al. (1989) J. Clin. Invest. 83:1015-1031; and Ignatius et al. (1987) Science 236:959-962.
Most recently, apoE has been implicated in Alzheimer's disease and cognitive performance. Saunders et al. (1993) Neurol. 43:1467-1472; Corder et al. (1993) Science 261:921-923; and Reed et al. (1994) Arch. Neurol. 51:1189-1192. ApoE4 is associated with the two characteristic neuropathologic lesions of Alzheimer's disease; extracellular neuritic plaques representing deposits of amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles representing filaments of hyperphosphorylated tau, a microtubule-associated protein. For review, see, McKhann et al. (1984) Neurol. 34:939-944; Selkoe (1991) Neuron 6:487-498; Crowther (1993) Curr. Opin. Struct. Biol. 3:202-206; Roses (1994) Curr. Neurol. 14:111-141; Weisgraber et al. (1994) Curr. Opin. Lipidol. 5:110-116; and Weisgraber et al. (1994) Curr. Opin. Struct. Biol. 4:507-515.
Alzheimer's disease is generally divided into three categories: early-onset familial disease (occurring before 60 years of age and linked to genes on chromosomes 21 and 14); late-onset familial disease; and sporadic late-onset disease. Both types of late-onset disease have recently been linked to chromosome 19 at the apoE locus. Other results suggest that apoE4 is directly linked to the severity of the disease in late-onset families. Roses (1994). Recently, cholesterol lowering drugs, the statins, have been suggested for use in treating Alzheimer's disease by lowering apoE4 levels. WO 95/06470.
The neurofibrillary tangles, which are paired helical filaments of hyperphosphorylated tau, accumulate in the cytoplasm of neurons. Tau is a microtubule-associated phosphoprotein which normally participates in microtubule assembly and stabilization; however, hyperphosphorylation impairs its ability to interact with microtubules. Increased binding of tau by apoE has been suggested as a treatment for Alzheimer's disease. WO 95/06456.
In vitro tau interacts with apoE3, but not with apoE4. Strittmatter et al. (1994) Exp. Neurol. 125:163-171. The interaction of apoE3 with tau may prevent its hyperphosphorylation, thus allowing it to function normally in stabilizing microtubular structure and function. In the presence of apoE4, tau could become hyperphosphorylated and thus inactive, which could promote the formation of neurofibrillary tangles.
ApoE4 has recently been associated with decreased learning ability and impaired memory. Helkala et al. (1995) Neurosci. Letts. 191:141-144. ApoE4 has been found to be a strong predictor of the outcome of patients designated as having memory impairment. Note that, apoE4 has been described as a risk factor, rather than a diagnostic. Peterson et al. (1995) JAMA 273:1274-1278; and Feskens et al. (1994) BMJ 309:1202-1206.
ApoE interacts with both the LDL receptor and the LRP and undoubtedly with other apoE-binding receptors on neurons. The LRP has been found to be increased after brain injury or glial cell conversion to necplasia. Lopes et al. (1994) FEBS Lett. 338:301-305. The LRP was previously identified as the macroglobulin receptor. Strickland et al. (1991) J. Biol. Chem. 266:13364-13369; and Borth (1992) FASEB J. 6:3345-3353. ApoE does not directly bind to the LRP but must first associate with cell surface heparin sulfate proteoglycans (HSPG). Mahley et al. (1991) Curr. Opin. Lipidol. 2:170-176; and Ji et al. (1994) J. Biol. Chem. 269:2764-2772. The LRP also binds a number of other ligands, including t-PA, I2-macroglobulin-protease complex, thrombospondin-1, Pseudomonas exotoxin A, the receptor associated protein (RAP) and lactoferrin. The LRP ligand binding sites have been at least partially described. Orth et al. (1994) J. Biol. Chem. 269:21117-21122; Godyna et al. (1995) J. Cell. Biol. 129:1403-1410; Kounnas et al. (1992) J. Biol. Chem. 267:12420-12423; Willnow et al. (1994) J. Cell Sci. 107:719-726; Meilinger et al. (1995) FEBS Lett. 360:70-74; Warshawsky et al. (1993) J. Biol. Chem. 268:22046-22054; and Willnow et al. (1994) J. Biol. Chem. 269:15827-15832.
It has previously been shown that incubation of dorsal root ganglion neurons in culture with β-VLDL alters the neurite growth of these cells compared to that of cells grown in media alone. Handelmann et al. (1992). In the presence of a source of lipid (β-VLDL or free cholesterol), neurite outgrowth is greatly enhanced, specifically due to extensive branching (with little or no increased neurite extension). When the β-VLDL was enriched with exogenous rabbit apoE (equivalent to human apoE3 with respect to the occurrence of a cysteine residue at position 112) enhanced neurite extension was seen. A lipid source appears to enhance membrane biosynthesis, whereas the addition of excess rabbit apoE with a lipid source results in long neuritic extensions and a trimming back of the branches. It has also been found that the inhibitory effect of apoE4 on neurite outgrowth is associated with microtubule polymerization, whereas apoE3 supports microtubule formation. Nathan et al. (1995) J. Biol. Chem. 270:19791-19799.
Neural plasticity, maintenance of existing or formation of new synaptic connections, is critical for normal brain function, including memory. This process can be compromised by various forms of stress, including, but not limited to, age, deposition of plaques and neurofibrillary tangles in Alzheimer's disease and oxygen deprivation. Interference with neuron remodeling can lead to impaired brain function or neurodegeneration of which dementia and Alzheimer's disease are extreme examples. In the case of Alzheimer's disease alone, approximately 4 million individuals are affected in the United States. With the aging of the population, this number is projected to triple in the next twenty years. The present health care cost of Alzheimer's disease is estimated at $90 billion per year in the United States alone. Delaying the average onset of this disease for even ten years would drastically reduce the financial burdens on society and the financial and emotional burdens of the families of these patients.
There are currently no effective therapies for arresting (and, more importantly, reversing) the impairment of central and peripheral nervous system function once an irreversible degenerative cascade begins. Likewise, there is no current therapy for restoration of normal, central and peripheral nervous system function when the induced stress has a less catastrophic or partially reversible effect compared to the dementias.
There is a need in the art for effective therapies for treating disorders associated with apoE4. The present invention addresses this need.